Anaerobic Migrating Blanket Reactor
Treatment of Low-Strength Wastewater at
Low Temperatures
Largus T. Angenent, Gouranga C. Banik, Shihwu Sung
ABSTRACT: The feasibility of the compartmentalized anaerobic
migrating blanket reactor (AMBR) was studied for the treatment of low-
strength soluble wastewater under low-temperature conditions. During
an operating period of 186 days, a 20-L AMBR was fed nonfat dry milk
substrate as a synthetic wastewater at low temperatures (15 and 20 C).
The concentration of the influent was constant at chemical oxygen
demand (COD) and 5-day biological oxygen demand (BOD5)
concentrations of 600 and 285 mg/L, respectively. The soluble COD
(SCOD) removal efficiency was 73% at the end of the operating period
(15 C) at a 4-hour hydraulic retention time (HRT), while the total COD
(TCOD) removal efficiency was 59%. At a 4-hour HRT, staged
conditions promoted complete removal of propionic acid in the final
compartments of the reactor. The specific methanogenic activity of
granules increased slowly until the end of the operating time, improving
the removal rate. Biomass was retained effectively, as evidenced by the
solids retention time (SRT) that was always greater than 50 days even
during step decreases of the reactor HRT from 12 hours to 4 hours. A
long SRT also promoted system stability during changes in flow, which
was observed by SCOD removal efficiencies staying greater than 70%.
During a hydraulic stress test, the HRT was reduced from 4 hours to 1
hour for one day (24 HRTs) in which volatile suspended solids (VSS) in
the effluent increased from an average background level of 8.7 g/d to 35
g/d and the SRT decreased from 50.5 days to 12.6 days. However,
mixed liquor volatile suspended solids concentration decreased only by
1 g/L, and hence a similar COD removal efficiency and biogas production
was found one day after the hydraulic stress (as compared to one day
before the hydraulic stress). Water Environ. Res., 73, 567 (2001).
KEYWORDS: anaerobic treatment, low-strength wastewater, low-tem-
perature conditions, compartmentalized reactor, granules, staged treatment,
anaerobic migrating blanket reactor, methanogenesis.
Introduction
Advantages of anaerobic pretreatment over aerobic treatment
for low-strength wastewater include decreased sludge production
and lower energy requirements, which result in decreased operat-
ing costs (Mergaert et al., 1992). Low-strength wastewater, such as
domestic and food-processing wastewater, is often discharged at
ambient temperatures (15 to 20 C) and heating the wastewater to
maintain mesophilic conditions (35 C) for anaerobic treatment
introduces large energy requirements. This would eliminate sav-
ings in operating cost for the anaerobic system completely. There-
fore, pretreatment of low-strength wastewater is more attractive
under low-temperature conditions. Recently, several laboratory-
and pilot-scale studies revealed promising results for high-rate
anaerobic treatment of low-strength wastewater at temperatures as
low as 3 to 5 C (Dague et al., 1998; Lettinga et al., 1999; and
Rebac et al., 1997). Most often an aerobic polishing (posttreat-
ment) step is needed after the anaerobic system (pretreatment) to
September/October 2001
meet effluent quality standards, such as for effluent nitrogen and
phosphorus levels (Rodrigues et al., 2001).
For full-scale treatment of low-strength wastewater, the upflow
anaerobic sludge blanket (UASB) reactor was used widely
(Hulshoff Pol et al., 1997, and Lettinga et al., 1993). The UASB
reactor consists of a single vessel with a hydraulic upflow pattern,
and hence a gassolids separator system and a feed-distribution
system are required to retain biomass and to distribute influent
evenly, respectively. Kato (1994) showed in laboratory-scale stud-
ies that the expanded granular sludge bed (EGSB) reactor, which
resembles a UASB reactor with a higher upflow velocity, was
more efficient compared with the UASB reactor for this type of
wastewater. Evidently, a higher mixing intensity in EGSB reactors
because of a higher upflow velocity decreased transport diffusion
limitations of substrate into the granules. Feasibility of this tech-
nology was confirmed with pilot-scale studies treating low-
strength malting wastewater at 13 C and 20 C (Rebac et al.,
1997).
Mechanically mixed, anaerobic sequencing batch reactors (AS-
BRs) were also thought to be an attractive option for the treatment
of low-strength, low-temperature wastewater, because increased
mixing conditions are combined with ideal conditions for biomass
settling and retention in the reactor. In addition, because of the
absence of a hydraulic upflow pattern, gassolids separator and
feed-distribution systems are superfluous, which simplified the
reactor configuration compared with UASB and EGSB systems.
Despite eliminating a hydraulic upflow pattern, the ASBR was able
to develop and grow granular biomass (Sung and Dague, 1995, and
Wirtz and Dague, 1996), which protects the strict anaerobes from
oxygen toxicity in low-strength, low-temperature wastewater treat-
ment (Kato, 1994). Studies by Dague et al. (1998) on ASBR
treatment of nonfat dry milk wastewater at temperatures ranging
from 5 to 25 C showed that solids retention times (SRTs) were
always exceeding 25 days and hence could offset the low growth
rates of biomass at these low temperatures. At a temperature of 5
C and a 6-hour hydraulic retention time (HRT), the ASBR still
achieved a total chemical oxygen demand (TCOD) removal effi-
ciency of 60%. An advantage of the ASBR technology was the
alternating higher and lower substrate levels (feast and famine
conditions) during the operating cycle, which resulted in increased
biomass settling and retention before effluent decanting and in-
creased substrate utilization rates just after feeding (Dague et al.,
1998). Increased substrate utilization is preferred, as for low-
strength wastewater substrate utilization rates are generally low,
dictated by Monod kinetics (Monod, 1949).
The kinetic advantage of relatively high substrate levels can also
567
Angenent et al.
be obtained with a continuously fed staged reactor configuration.
Lettinga et al. (1999), for example, used two EGSB reactors in
series to gain from higher substrate concentrations in the first
EGSB reactor, when feeding a low-strength volatile fatty acid
(VFA) synthetic wastewater at a 1.5-hour HRT and a temperature
of 8 C. These researchers found also that staging was beneficial
for the following reasons: (1) more favorable conditions for pro-
pionic acid degradation in the staged-reactor configuration because
of low acetic acid and hydrogen levels in the second stage; and (2)
relative high levels of bacteria in the first stage of the system
(while methanogens were still present) and relative high levels of
methanogens in the second stage (biomass staging), which im-
proved biodegradation kinetics due to each major group of micro-
organisms existing in an optimal environment (Lettinga et al.,
1999, and van Lier et al., 1997).
Another continuously fed staged reactor, the anaerobic migrat-
ing blanket reactor (AMBR) was developed, because there is still
a need for simple and cost-effective high-rate anaerobic treatment
systems for small and medium sized communities and industries,
especially for treating low-strength wastewater (Hulshoff Pol et
al., 1997). The AMBR utilizes the advantages of the ASBR, such
as mechanical mixing, biomass retention, a simple design (no
gassolids separation and feed-distribution systems required be-
cause of the absence of a hydraulic upflow pattern), and granula-
tion (Angenent and Sung, 2001). In addition, the HRT in a con-
tinuously fed AMBR can be shortened (and thus the reactor
volume can be decreased) compared with the batch-fed ASBR.
Relatively long HRTs due to physical limitations of the system (a
combination of a batch-fed operation and a relatively tall granular
blanket) are required for the ASBR, because a reduction of the
6-hour HRT for ASBRs treating low-strength wastewater was not
anticipated (Dague et al., 1998).
Because of the staged and continuously fed configuration of the
AMBR (and thus a potential for short HRTs) in combination with
a simple design, the feasibility of the AMBR system for the
treatment of highly soluble, low-strength wastewater was studied.
The AMBR treatment was evaluated under low-temperature con-
ditions (15 C) by monitoring reactor performance over a period of
6 months, during which the HRT was reduced from 12 hours to 4
hours in a stepwise manner. Biomass retention, granular size, and
methanogenic activity of granules were also monitored during this
period. In addition, a sudden drop of HRT from 4 hours to 1 hour
was introduced to study the behavior of the AMBR under hydrau-
lic-stress conditions, which are common for low-strength waste-
water.
Materials and Methods
Anaerobic Migrating Blanket Reactor. The AMBR, with an
active volume of 20 L (inside width: 140 mm; inside length: 580
mm), was divided into four compartments (Figure 1). Baffles were
placed between the compartments to reduce short-circuiting of
substrate. The space between a baffle and the inside wall was 10
mm to prevent clogging by large granules. The temperature of the
AMBR was kept constant at 20 1 C for the first 87 days of
operation. At day 88, the temperature was decreased to 15 1 C,
and precooling of influent became necessary. The flow over the
horizontal plane of the reactor was reversed once a day. A daily
change in flow direction was chosen to prevent a pH drop due to
VFA buildup in the initial compartment and to prevent unequal
biomass levels due to anticipated biomass migration between
compartments.
568
Figure 1Schematic diagram of AMBR (EBS effluent
baffle system).
Before the flow was reversed, the second compartment was step
fed for 4 hours (after termination of feeding the initial compart-
ment) to prevent a breakthrough of substrate. Sufficient contact
between substrate and biomass was maintained using intermittent,
gentle mixing. Mixers (model 5vb, EMI Inc., Clinton, Connecti-
cut) were mounted with paddles to ensure gentle mixing. At a
rotational speed of 60 r/min, these paddles produced a root mean
square velocity gradient (G) of 77 s1 in a 5-L compartment, as
determined by a rotating torque meter (Bex-O-Meter, model 38,
The Bex Company, San Fransisco, California) described in Sajjad
and Cleasby (1995). The compartments were mixed equally for 10
seconds every 4 minutes at 60 r/min for the first 155 days of
operation. At day 156, the mixing frequency was doubled to once
every 2 minutes in the three initial compartments, and the final
compartment was mixed every 4 minutes to prevent excessive
biomass loss. Increasing the mixing frequency of the initial com-
partments was implemented to decrease transport limitation and
short-circuiting of substrate at higher organic loading rates. All
pumps used were Masterflex pumps (Cole Parmer Instrument Co.,
Chicago, Illinois).
The gas collection systems consisted of an observation bottle, a
gas sampling port, and a wet-test gas meter (GCA, Precision
Scientific, Chicago, Illinois). The biogas was directly discharged
from the reactor to the gas collection system. A water head was
installed on the effluent tubes to prevent biogas from escaping
directly through the effluent ports. Timers (ChronTrol Corpora-
tion, San Diego, California) regulated the operation. An effluent
baffle system was placed in front of the effluent ports to prevent
floating granules from leaving with the effluent (Figure 1).
Inoculum. Granular inoculum was obtained from laboratory-
scale ASBRs fed with the same substrate (Dague et al., 1998) and
was stored for 3 months at 5 1 C and for 1 month at 20 1 C
before operation. These granules were acclimated to psychrophilic
temperatures as low as 5 1 C. At start-up, the mixed liquor
volatile suspended solids (MLVSS), which is an indication of the
amount of biomass in the reactor, was 20 g/L.
Substrate. A concentrated substrate stock solution, consisting
of nonfat dry milk (962 mg/g chemical oxygen demand [COD]),
sodium bicarbonate (962 mg/g feed COD) and trace elements
(ferrous chloride, FeCl24H2O: 17.1 mg/g COD, nickel chloride,
NiCl26H2O: 1.9 mg/g COD, cobaltous chloride, CoCl26H2O: 1.9
mg/g COD, manganese chloride, MnCl24H2O: 1.7 mg/g COD,
and zinc chloride, ZnCl2: 1.3 mg/g COD), was stored at 4 1 C
and was mixed during feeding to obtain a constant loading rate.
Makeup water was added to the concentrated substrate just before
introduction to the reactor to obtain a COD and 5-day biochemical
oxygen demand (BOD5) concentration of 600 mg/L and 285 mg/L,
respectively (the BOD5/COD ratio was 0.49). The soluble fraction
Water Environment Research, Volume 73, Number 5

of COD (SCOD) of the nonfat dry milk substrate solution was on
average 8.4% smaller than the TCOD fraction. The sulfate con-
centration of the influent (mainly from the city of Ames, Iowa, tap
water) was approximately 110 mg/L (COD/sulfate ratio was 5.5).
Analyses. Concentrations of nitrogen, methane, and carbon
dioxide in the headspaces of the AMBR and activity bottles were
measured using a gas chromatograph (model 350, Gow-Mac In-
struments, Co., Bridgewater, New Jersey) with a thermal conduc-
tivity detector (column: 1.7 m x 3 mm stainless steel Poropack Q
0.177/0.149 mm [80/100 mesh]; carrier gas: helium). The individ-
ual VFA levels were measured with an ion chromatograph (model
DX-500, Dionex, Sunnyvale, California) containing a CD 20 con-
ductivity detector and an anion micromembrane suppressor (col-
umn: Ion Pac ICE-As1; eluant: 0.8 to 1.0 mM heptafluorobutyric
acid). For VFA analyses, samples were first acidified with hydro-
chloric acid. The total alkalinity, total VFAs, TCOD, SCOD, total
suspended solids (TSS), volatile suspended solids (VSS), and
sludge volume index tests were performed according to procedures
described in Standard Methods (APHA et al., 1995). Effluent
samples were taken at the midpoint of the time interval between
reversals of flow.
Biomass Characteristics. The specific methanogenic activity of
biomass was assessed at 35 1 C using the headspace method These loading rates at a temperature of 20 C were possible,
according to tests described by Rinzema et al. (1988). To analyze
the change in size of the granules during the operational time, the
arithmetic mean diameter [Sum (d/n), where d is the granule
diameter and n is the number of granules] and area-weighted mean
diameter [Sum(d3)/Sum(d2)] were calculated by automated image
analysis. Mixed liquor samples were collected six times through-
out the course of the study and diluted to obtain fully suspended
granular biomass samples for image analysis. Next, 1.75 mL of
this sample was added to a specially prepared slide consisting of
two, 3-mm thick glass sheets cemented together, with a 25-mm
diameter hole in the top sheet. The image analysis setup contained
a black and white video camera (series 68, Dage-MTI, Michigan
City, Indiana), a microscope (model SZH, Olympus, Melville,
New York), and a personal computer with Quartz PCI Imaging
software (Quartz Imaging Corporation, Vancouver, British Colum-
bia, Canada). Some manual editing of the image was necessary to
separate adjacent granules. Particles smaller than 0.1 mm were not
included in the calculations of the size distribution (Grotenhuis et
al., 1991).
The biomass migration rate was determined by the decline in
MLVSS from the initial compartment during the time of feeding.
The food/microorganism (F/M) ratio was calculated by dividing
the mass of COD fed per day with the total VSS in the reactor. The
SRT was determined by dividing the total VSS in the reactor with
the daily loss of VSS in the effluent.
Assessment of Reactor Performance. To obtain information
on the performance of the system, COD removal efficiencies were
based on COD concentrations measured in the influent and effluent
and methane production. The volumetric loading rate (VLR) was
calculated as the mass of COD fed to the system per reactor
volume per day (g COD/Ld). Data for the SCOD and TCOD
removal efficiencies were obtained by measuring the COD con-
centrations of the feed and the effluent. Effluent samples and
samples from individual compartments were taken at the midpoint
of the time interval between two reversals of flow. Methane
production was determined as follows. First, the biogas production
(gas meter) was corrected to standard temperature and pressure
(STP). Next, the standard methane production rate (SMPR) was
September/October 2001
Angenent et al.
obtained after dividing the biogas production with the wet volume
of the reactor and converting it to the methane percentage that was
present in the biogas. Therefore, the SMPR was expressed as
volume of methane production per reactor volume per day (L/Ld).
The methane that left the reactor in a soluble form with the effluent
was then calculated. First, the solubility of methane in water at 15
or 20 C was estimated using Henrys law (Perry et al., 1997).
Second, the methane quantity per day that left the reactor through
its effluent was calculated and was corrected to STP and converted
to SMPR. Theoretically, 0.35 L of methane has 1 g of COD, which
also means 1 g of COD can potentially generate 0.35 L of methane
at STP (when biomass growth is ignored). Hence, the methane-
based COD (MCOD) removal efficiency can be calculated by the
total SMPR (methane that left the reactor through the gas meter
and the effluent) using the following formula:
SMPR
MCOD removal efficiency, % (100)
(VLR)(0.35)
Results and Discussion
Operating Conditions. At the start of the operating period, the
reactor was fed at a 12-hour HRT and a VLR of 1.25 g COD/Ld.
because the inoculum was already acclimated to similar environ-
mental conditions. The concentration of the influent was kept
constant at 600 mg COD/L (as TCOD) during the entire operating
period and decreasing the HRT resulted in an increase in the VLR.
At the end of operating time, the HRT was 4 hours, which resulted
in a VLR of 3.5 g COD/Ld (Figure 2a). The operating length of
each different HRT was chosen based on TCOD, SCOD, and
MCOD removal performance. If two or more data points were
within 5% margin after the system was operated for at least 10
HRTs, a quasi steady state was determined. True steady-state
conditions were not achieved at the HRTs of 12, 8, and 4 hours,
because the MLVSS and SRT were still increasing slowly while
the HRT was decreased again (Figure 2c). It needs to be empha-
sized that the AMBR is a dynamic process in which substrate
concentrations and environmental conditions change over time
because of reversing the flow.
Figure 2 illustrates the operating conditions during the operating
period. Biomass levels in the reactor stayed at approximately 23
g/L during this period (Figure 2b). Meanwhile, VSS losses with
the effluent oscillated around a mean of 4.4 g/d (standard error,
SE 1.9; number of datapoints, n 18) during the operating
period (not including the hydraulic stress data). The oscillation
pattern was the result of temporary solids loss in the effluent after
a temperature decrease at day 88 and after the increased flows due
to HRT changes. Even during the temporary biomass loss in-
creases, biomass retention was high and the SRT remained greater
than 50 days. After a period of acclimation, VSS levels in the
effluent decreased and the SRT increased to values greater than
100 days. The HRT could then be decreased again (by increasing
the flow), which resulted in temporarily lower SRTs of 50 days
(Figure 2c). Because the MLVSS levels remained constant, F:M
during the operating period increased from 0.05 to 0.18 g COD/g
VSSd, depending on VLR (Figure 2c). Meanwhile, pH levels in
the initial compartment measured at the midpoint between rever-
sals of flow (12 hours of feeding the initial compartment) were
constant over the operating period with a mean of 6.7 (SE 0.1;
n 30). The alkalinity in the effluent remained constant as well
(mean 0.53 g/L as calcium carbonate; SE 0.04; n 18).
569
Angenent et al.

Therefore, the increase in F:M showed no decrease in pH level in

the initial compartment, which indicates that stable conditions
prevailed in the reactor.
Anaerobic Migrating Blanket Reactor Performance. Figure
3 illustrates the reactor performance over the operating period. At
a 12-hour HRT, biogas production (measured with the gas meter)
reached a plateau within a month of feeding the reactor at a
temperature of 20 C (Figure 3a). At this period (days 24 to 43),
the AMBR achieved SCOD, TCOD, and MCOD removal efficien-
cies of 93.9% (SE 1.8; n 3; average effluent SCOD concen-
tration, eSCOD 34.6 mg/L), 81.3% (SE 5.0; n 3; average
effluent TCOD concentration, eTCOD 105.5 mg/L), and 61.0%
(SE 7.9; n 20), respectively (Figure 3b). The MCOD removal
efficiencies were lower than the TCOD removal efficiencies pri-
marily because of biomass accumulation in the reactor (Figure 2b).
The lower MCOD removal efficiency can also be partly explained

Figure 3Reactor performance: (a) biogas and methane

production, (b) COD removal efficiency, and (c) biomass
activity and granular size. Results during the hydraulic
stress test are indicated by peak flow.

by sulfide formation by sulfate-reducing bacteria and the subse-

Figure 2Operating conditions: (a) loading conditions,
(b) biomass concentrations in reactor and effluent, and
(c) SRT and F:M. Results during the hydraulic stress test
are indicated by peak flow.
quent sulfide loss, and thus COD loss, through stripping of hydro-
gen sulfide or precipitation of metal sulfides. A theoretical maxi-
mum of 10% of the SCOD removed can be attributed to this
phenomenon when 100 mg/L sulfate was reduced and all sulfide
was stripped or precipitated. An increased VLR resulting from the
HRT decrease from 12 hours to 8 hours increased the biogas
production slowly until day 78. Feeding was terminated for 2 days
from day 78, which resulted in a reduced biogas production. After
feeding the system again for 7 days, the biogas production reached
a level similar to that before the termination of feed.
Reducing the operating temperature from 20 C to 15 C on day
88, decreased the biogas production (as measured with the gas
meter) from approximately 8 L/d to 6 L/d and the SMPR (gas
meter) from approximately 0.3 L/Ld to 0.2 L/Ld (Figure 3a). The
increased level of soluble methane in the effluent at 15 C com-
pared with 20 C did not correct entirely for the decrease in
measured methane and biogas production. After decreasing the

570 Water Environment Research, Volume 73, Number 5


temperature, the SMPR for the soluble methane fraction accounted
for one third of the total SMPR at a temperature of 15 C (Figure
3a). The biogas production remained at a similar level for the rest
of the 8-hour HRT period (days 94 to 108) during which the
SCOD, TCOD, and MCOD removal efficiencies were 82.1%
(SE 1.0; n 3; eSCOD 96.3 mg/L), 70.3% (SE 3.0; n typical activated-sludge growth yields are greater than (0.6 g
3; eTCOD 159.8 mg/L), and 53.4% (SE 7.3; n 13),
respectively. These removal efficiencies decreased further when
the HRT was reduced from 8 hours to 6 hours. At the end of the
6-hour HRT period (days 157 to 165), the biogas production
increased slowly to approximately 7 L/d (as measured with the gas
meter). During this period, the AMBR achieved SCOD, TCOD,
and MCOD removal efficiencies of 75.9% (n 2; eSCOD
136.1 mg/L), 62.1% (n 2; eTCOD 213.7 mg/L), and 42.8%
(SE 2.1; n 9), respectively, which were lower than those at the
8-hour HRT and temperature of 15 C (Figure 3b).
The biogas production increased to approximately 8 L/d (gas
meter) when the HRT was further decreased to 4 hours, indicating
that the system was increasing its removal rates. An increase in
removal rate was also determined by the specific methanogenic
activity of the granules, which increased slowly for the final 100
days of operation (Figure 3c). The increase in removal rate resulted
in only slightly lower COD removal efficiencies at a 4-hour HRT
compared to the 6-hour HRT. The SCOD, TCOD, and MCOD
removal efficiencies for the two data points (days 172 and 184) at
a 4-hour HRT before the hydraulic stress test were 73.0% (n 2;
eSCOD 163.9 mg/L), 59.2% (n 2; eTCOD 248.4 mg/L),
and 35.6% (SE 1.7; n 11), respectively. The lower SCOD
removal efficiencies were a result of an increase in the VFA
concentrations of the effluent from 30 mg/L as acetic acid (n 2)
to 60 mg/L (n 2) during the HRT decrease.
The operating performance showed an SCOD removal effi-
ciency greater than 70% throughout the operating period, while the
TCOD removal efficiency remained at approximately 60% for a
4-hour HRT. These values did not dramatically decrease after HRT
changes, and hence the system showed stability after flow in-
creases. During each period of a 12, 8, and 6-hour HRT, the
performance of the AMBR improved slightly. Therefore, the per-
formance at a 4-hour HRT was expected to have improved at a
longer operating period, because the biogas production and spe-
cific methanogenic activity of the granules were increasing slowly
at the end of the operating time. Dague et al. (1998) performed
similar studies with ASBRs fed with a similar nonfat dry milk
substrate. At a 6-hour HRT and a temperature of 15 C, these
workers found an SCOD removal efficiency of 81.5%. A slightly
lower SCOD removal efficiency of 75.9% at similar conditions
was achieved during the current study. Although the ASBR
achieved a higher SCOD removal efficiency, the HRT could not be
reduced from 6 hours to 4 hours because of physical limitations of
the batch-fed system. In contrast, the HRT of the AMBR was
reduced to 4 hours and was even decreased to 1 hour during the
hydraulic stress test.
Pretreatment of raw domestic wastewater by a UASB system at
ambient temperatures was studied by Barbosa and Sant Anna
(1989). Although solids were present in their wastewater, the
BOD5 and COD concentrations were 357 and 627 mg/L, respec-
tively, which were close to those of the current study (285 and 600
mg/L, respectively). Similar to this previous study, current results
suggest that the AMBR as a pretreatment step can be effective in
removing COD, and thus can reduce energy requirements and add
stability to an aerobic posttreatment facility. Moreover, sludge
September/October 2001
Angenent et al.
production can be reduced greatly, as the data suggest a true
growth yield of 0.16 g biomass COD formed/g SCOD removed
over the final three data points at a 6-hour HRT (15 C). This is in
agreement with ASBR data that showed a true growth yield of
0.145 g COD/g COD at 15 C (Banik et al., 1998). In contrast,
COD/g COD) (Grady et al., 1999). In contrast to the procedures
used by Barbosa and Sant Anna (1989), the AMBR of the current
study was operated with a highly soluble synthetic wastewater. For
nonsettled domestic wastewater that contains relatively high levels
of suspended solids, the AMBR can be operated at a longer HRT
to facilitate primary solids separation and the possibility to main-
tain flocculent biomass instead of granular biomass to promote
suspended and colloidal solids destruction. For this type of waste-
water, hydrolysis and acidogenesis rather than methanogenesis
become the rate-limiting steps, and hence to provide for solids
destruction, the entrapment of suspended solids and long SRTs
become important (Switzenbaum and Grady, 1986). An AMBR
operated with (partly) flocculent biomass may provide for suffi-
cient biomass and solids retention, because this study with granular
biomass showed an SRT that was longer than 100 days during
pseudo-steady-state conditions. However, further studies are
needed to elucidate the feasibility for AMBR treatment of non-
settled domestic wastewater.
Besides reducing nutrient levels in the effluent, posttreatment of
anaerobic reactor effluent is needed to reduce the mean SCOD
concentrations of 106 mg/L (SE 50; n 19) as found in this
study. The soluble organic material in the effluent consisted of
VFAs with a mean total concentration of 39 mg/L as acetate (SE
14; n 21). The remaining soluble organic material may have
partly consisted of soluble microbial products (Langenhoff and
Stuckey, 2000). Most soluble microbial products are not, or
slowly, biodegradable and, therefore, are not measured with BOD5
tests. Hence, BOD5 measurements and removal efficiencies would
have given an additional evaluation of the performance of the
AMBR, especially as the nonfat dry milk substrate was not fully
degraded under aerobic conditions within 5 days (BOD5/COD
ratio of 0.49).
Biomass Characteristics. An increase in the size of the gran-
ules over time is illustrated in Figure 3c. At the end of the
operating period, the arithmetic and area-weighted mean diameter
were 1.1 and 3.0 mm, respectively. Also, the area distribution of
the granules in Figure 4 shows an increase in granular diameter
over the operating time, during which the graph at day 185 had
shifted to the right compared with the graph of the seed granules
and the graph at day 105. At day 185, 40% of the projected
granular area was due to particles between 3.16 and 5.62 mm in
diameter. For the seed sludge no particles with a diameter between
3.16 and 5.62 mm were found (Figure 4). A selection for larger
granules occurred because of growth and an increasing washing
out of smaller biomass at higher flows. It needs to be realized that
selection of larger granules occurred despite the absence of a
hydraulic upflow pattern. At the end of the operation, the migration
rate of large granules was virtually zero because of their good
settling characteristics (sludge volume index was 18.7 mL/g) and
a compartmentalized configuration of the AMBR. This absence of
migration became ubiquitous because a difference in granular
structure was observed between the compartments. The surfaces of
granules from the outside compartments showed an abundance of
white colonies (75 m in diameter), consisting of microorganisms
with a different morphology (microscopic observations of the
571
Angenent et al.

colonies showed a homogeneous population), while the granules

from the inside compartments continued to have a smooth black
surface. Because of high concentrations of sulfate in the influent
(110 mg/L), these microcolonies, presumably, consisted of sulfate-
reducing bacteria as was also described by Sekiguchi et al. (1999),
who discovered colonies consisting of Desulfobulbus spp. on gran-
ules by using fluorescence in situ hybridization.
The specific methanogenic activity of biomass for the outside
compartments from the AMBR was 0.53 g COD/g VSSd (stan-
dard deviation, SD 0.01; number of measurements, m 2) and
was 0.58 g COD/g VSSd (SD 0.01; m 3) for the inside
compartments. Hence, at the end of the study a small difference in
specific methanogenic activity was found between the granules of
the inside and outside compartments (t-test: 95% significance
level), which resulted in biomass staging. This nomenclature
indicates that different microbial communities developed over the
length of a plug-flow reactor or in a sequence of compartments.
For example, biomass staging in a two-stage EGSB reactor fed
with partly acidified substrate at temperatures as low as 8 C,
resulted in a dominant acidogenic population in the first stage and
dominant acetogenic and methanogenic populations in the second
stage (van Lier et al., 1997). Results of the current study suggest,
however, a much smaller difference in biomass between the com-
partments. The reversing flow scheme is the most likely reason
why biomass staging was limited for the AMBR compared with
the unidirectionally fed, two-stage EGSB system. The above given
specific methanogenic activity was measured at 35 C, which
verified that mesophilic methanogens remain active over the op-
erating period despite low-temperature conditions, as was also
found by Lettinga et al. (1999), Banik et al. (1997), and Langen-
hoff and Stuckey (2000).
Hydraulic Stress. At day 185, the HRT was decreased from 4
hours to 1 hour to study the effect of a temporary increase in
wastewater flow (hydraulic stress). During this 24-hour period (24
HRTs), a total of 480 L of feed was applied to both flow directions
(240 L was fed to each direction for 12 hours). One day before the
hydraulic stress test, at day 184, staged environmental conditions
in the AMBR were apparent with relatively high concentrations of
acetic acid and SCOD in the initial compartment (compartment 1)
and low concentrations in the final compartment (Figure 5a). The Figure 5Volatile fatty acid and SCOD concentration at
appearance of formic acid and propionic acid in the initial com- the midpoint between intervals of flow per compartment:
partment indicated the occurrence of substrate staging. In this (a) t 184 days and general operating conditions at a
4-hour HRT; (b) t 185 days and hydraulic stress at a
1-hour HRT; and (c) t 186 days and day after hydraulic
stress at a 4-hour HRT.

paper, substrate staging indicates that different substrate levels

Figure 4 Area distribution of granules from the start,
middle, and end of the operating period.
(and thus environmental conditions) are present over time or over
a spatial arrangement of the reactor configuration, regardless of
whether biomass staging occurs. Propionic acid concentrations that
were below the detection limit illustrated favorable propionic acid
degradation in compartments 2 to 4. Grobicki and Stuckey (1992),
van Lier et al. (1996), and Langenhoff and Stuckey (2000) showed
that for compartmentalized anaerobic configurations the actual
number of compartments correlates closely with an equal number
of perfectly mixed compartments, and that, as such, plug-flow
conditions were approached. The resulting SCOD and VFA con-
centration gradients at a 4-hour HRT were used as a baseline to
compare reactor performances during and after the hydraulic
stress.

572 Water Environment Research, Volume 73, Number 5


The 4-hour to 1-hour HRT drop at day 185 increased the VLR
from 3.5 g COD/Ld to 14.4 g COD/Ld (COD level of the feed
was kept constant). Consequently, the F/M ratio was increased
from 0.18 to 0.72 g COD/g VSSd (Figure 2c). The SCOD, TCOD,
and MCOD removal efficiencies decreased to 39.3% (eSCOD
384.3 mg/L), 30.3% (eTCOD 441.2 mg/L), and 14.8% (SE
0.8; n 4), respectively, while the VFA concentration of the
effluent increased to 110 mg/L as acetic acid (Figure 3b). Despite
lower removal efficiencies, the AMBR removed more SCOD
during than before the hydraulic stress test (123.4 and 58.5 g/d,
respectively). The hydraulic stress did not upset the reactor in
terms of a pH drop in the initial compartment, but reactor perfor-
mance (COD removal efficiencies) was deteriorated severely. Sub-
strate staging that was apparent at a 4-hour HRT was lost com-
pletely at day 185 (Figure 5b). For example, the acetic acid
concentration in compartment 1 was lower than that in compart-
ments 2 to 4. Moreover, propionic acid levels that were lower than
the detection limit in compartments 2 to 4 at a 4-hour HRT, had
reached a level as high as 22 mg/L during the hydraulic stress.
Clearly, a shift in degradation of nonfat dry milk substrate had
occurred from the initial compartment to the middle and final
compartments because of the flow increase from 120 to 480 L/d.
The biomass migration rate during the hydraulic stress increased
from virtually zero to approximately 13.2 g VSS/Ld, and hence
more granules were migrating through the system. Also, the wash
out of biomass with the effluent increased from 8.7 to 35 g VSS/d
(Figure 2b), which decreased the SRT from 50.6 to 12.6 days
(Figure 2c). However, after 1 day of feeding the AMBR at a 1-hour
HRT, the MLVSS had decreased only by 1 g/L. Because of the
retention of biomass in the system, the reactor performed almost
similarly the day after the hydraulic stress as compared to the day
before the hydraulic stress, with SCOD, TCOD, and MCOD re-
moval efficiencies of 69.6% (eSCOD 168.1 mg/L), 49.2%
(eTCOD 280.5), and 32.8%, respectively (Figure 3b). Figure 5c
illustrates that the AMBR showed substrate staging at day 186
similar to that before the hydraulic stress. Only slightly elevated
acetic and propionic acid concentrations of approximately 10 mg/L
in compartments 2 to 4 indicated that the system had been stressed
(Figure 5c). Studies with an alternative compartmentalized anaer-
obic system, called the anaerobic baffled reactor, also showed a
compartmentalized system to be very stable during large changes
in flow (Nachaiyasit and Stuckey, 1997). The anaerobic baffled
reactor recovered to its baseline performance shortly after the
period of hydraulic stress ended. Hence, anaerobic systems with a
configuration that promotes high biomass retention are required in
off-setting hydraulic stress. This is especially important for low-
strength wastewater at low-temperature conditions, because of the
low biomass growth rate.
Conclusions
A laboratory-scale AMBR, which consisted of four compart-
ments and was fed with a nonfat dry milk synthetic wastewater at
a concentration of 600 mg COD/L (BOD5 of 285 mg/L) at a
temperature of 15 C, achieved SCOD, TCOD, and MCOD re-
moval efficiencies of 73%, 59%, and 36%, respectively, at a
4-hour HRT. The reactor removal rate improved over the operating
time, during which the specific methanogenic activity of the gran-
ules increased slowly. The improvement in removal rate was
possible because of long biomass SRTs in the AMBR resulting
from effective granular retention. Biomass retention also promoted
September/October 2001
Angenent et al.
stability of the system during changes in flow, as was demonstrated
during a temporary drop in HRT from 4 hours to 1 hour.
The results of this study indicate that the AMBR is an attractive
option for treating highly soluble industrial and domestic low-
strength wastewater at low-temperature conditions, because the
AMBR showed high biomass retention and stability during
changes in flow. Moreover, at a 4-hour HRT it showed substrate
staging with relatively high SCOD and acetic acid concentrations
in the initial compartment and low SCOD and acetic acid concen-
trations in the final compartment. These conditions were beneficial
in promoting a complete removal of propionic acid in the final
compartments of the reactor. We did not study the dynamic be-
havior of the AMBR because of the flow reversals over the
horizontal plane of the reactor and the possible advantages this
might have on reactor stability. Also, we did not optimize the
frequency of reversing the flow over the horizontal plane of the
reactor. Hence, further studies should include optimization of the
AMBR operation, while feeding real domestic or low-strength
industrial wastewater.
Acknowledgments
Credits. This research was made possible by a grant from the
U.S. Department of Agriculture (Washington, D.C.), through the
Iowa Biotechnology Byproducts Consortia (Ames, Iowa). The
authors acknowledge Richard Dague, who initiated anaerobic re-
search with low-strength wastewater at Iowa State University
(Ames) and Warren Straszheim (Civil and Construction Engineer-
ing Material Analyses Research Laboratory, Iowa State Univer-
sity) for his assistance with image analyses. This paper was pre-
sented in part at WEFTEC 2000, the Water Environment
Federations 73rd Annual Conference and Exposition held in Ana-
heim, California, October 14 18, 2000.
Authors. At the time of this work, all authors were in the
Department of Civil and Construction Engineering at Iowa State
University. Largus T. Angenent is currently a postdoctoral re-
search associate in the Department of Civil, Environmental, and
Architectural Engineering, University of Colorado at Boulder;
Gouranga C. Banik is an assistant professor in the construction
department at Southern Polytechnic State University (Atlanta,
Georgia); and Shihwu Sung is an assistant professor in the De-
partment of Civil and Construction Engineering, Iowa State Uni-
versity. Correspondence should be addressed to Largus T. Ange-
nent, Department of Civil, Environmental and Architectural
Engineering, University of Colorado at Boulder, Campus Box 428,
Boulder, CO 80309.
Submitted for publication November 16, 2000; revised manu-
script submitted May 14, 2001; accepted for publication August
28, 2001
The Deadline to submit Discussions of this paper is February
15, 2002.
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